GSM7178538: Streptomyces coelicolor A3(2) grown in liquid SMM medium, control, 0 min, total RNA, biol replicate 1; Streptomyces coelicolor A3(2); RNA-Seq
Polysome and monosome associated RNA was extracted by mixing 3 volumes of Trizol-LS (Invitrogen 10296028) to 1 volume of lysate by vortex mixer for 15 seconds and incubating 5 minutes at room temperature. 200 µl chloroform were added and samples were mixed by vortex mixer for 15 seconds, incubated at room temperature for 5 minutes and centrifuged to separate the phases. Upper phase was retained and the RNA precipitated by adding 800 µl of isopropanol and 1µl of glycoblue (Invitrogen AM9516) and the RNA was precipitated by incubation at -80°C for 1 hour. Samples were centrifuged at 9,838 RCF / 4°C for 1 hour and the supernatant discarded, the RNA pellet washed twice in 1 ml ice-cold 75% ethanol and then dissolved in nuclease-free water. For total RNA extraction (transcriptome analysis),  bacterial cultures were treated with RNA protect bacteria reagent ( Qiagen #76506) and incubated for 10 min at room temperature. Cultures were centrifuged and the pellets were stored at -80C. Pellets from 10 ml (time=0 reference) or 40ml (cold-shock or control samples) of bacterial cultures were resuspended in 200 µl of lysozyme dissolved in TE buffer at a concentration of 0.015 g per ml and incubated at room temperature for 15 min. 600 µl of RNeasy Lysis Buffer (RLT buffer, Qiagen #79216) containing 10 µl/ml β-mercaptoethanol was added and the mixture mixed by vortex mixer then transferred to a 2 ml microfuge tube containing 5 mm stainless steel beads (Qiagen 69989) and placed in a Tissuelyser LT (Qiagen #85600) for efficient mechanical cell disruption / homogenisation for 2 min at 30 rpm. The suspension was then centrifuged at 16,627 RCF for 2 min, the supernatant was recovered and its volume estimated. One volume of acid pH 4.5 phenol:chloroform:isoamyl alcohol (25:24:1, Fisher #AM9720) was added (for the first replicate of the solid media cultures neutral pH phenol:chloroform:isoamyl alcohol(25:24:1, Sigma #77617 was substituted)), and the mixture mixed by vortex mixer for 30 seconds, then centrifuged at 4°C for 5 min at 16,627 RCF. The upper phase containing the nucleic acids was removed and retained leaving behind the proteins in the interface, and the phenol/chloroform extraction repeated twice more. Then one volume of chloroform was added, the mixture mixed by vortex mixer then centrifuged at 4°C for 2 min at 16,627 RCF, and the upper phase was again transferred to a fresh RNase-free microfuge tube. From this point onward the standard manufacturer's instructions were followed for the miRNAeasy RNA extraction kit (Qiagen #217004). The total RNA was then eluted in 30 µl of nuclease free water and stored at -80°C. Genomic DNA was removed using the Turbo DNA free kit (Invitrogen AM1907). Total RNA and monosome and polysome associated RNA were depleted of rRNA by using the NEBNext Core Reagent Set (NEB #E7865) with custom DNA probes designed to cover all S. coelicolor rRNA genes plus rnpB, ssrA and srp, and library constructions were carried out using the NEB directional RNAseq kit (NEB #E7760).